Genetic engineering part 3

Continuation of genetic engineering (part 2): Vector

The sticky ends are generated by the same enzyme on vector as well as the target DNA are complementary and hence are cohesive. The sticky ends of the cleaved DNA segment cohere with those of the vector, thus the cut DNA sequence can now be introduced into the plasmid. The cut ends are joined by DNA ligase enzyme and the introduced gene becomes a part of the plasmid. Ligase is an enzyme that covalently joins the sugar-phosphate backbone of bases together. Ligase will join either "sticky" ends or "blunt" ends, but it is more efficient at closing sticky ends. The process of introducing foreign gene into a vector is called as cloning and the plasmid containing a cloned gene is called chimera.

Illustration of cloning click to enlarge


If the foreign DNA and the cloning vector does not have a common restriction site at the required position, they may still be spliced through the use of terminal deoxynucleotidyl transferase enzyme. This mammalian enzyme adds nucleotides to 3’-terminal OH group of a DNA chain. It is the only known DNA polymerase that does not require a template. Using this enzyme and dTTP, long poly(dT) tails are build up at the 3’ end of DNA sequence to be cloned. The cloning vector is also enzymatically cleaved at a specific site and 3’ ends of the cleavage sites are extended with poly (dA) tails. The complimentary homopolymer tails are annealed and the strands are joined by DNA ligase. Since the foreign DNA lacks any restriction site, it becomes difficult to recover the insert from the vector.

Illustration on using terminal deoxynucleotidyl transferase enzyme Click to enlarge



Another method to over this problem is to use specially designed palindromic “linker” that are appended to either ends of the DNA insert. This linker is a chemically synthesized DNA fragment that has the same restriction site present in the vector. The linkers are attached to inserts by blunt end ligation with T4 ligase. They are then cleaved with appropriate restriction enzyme resulting in generation of sticky ends at either sides of the insert. The sticky ends of the vector and those of target DNA sequence (with linker) cohere. The strands are annealed and ligated by DNA ligase enzyme.

Illustration on using palindromic linker Click to enlarge


The wild λ bacteriophage has a genome of 48.5 kb, of which the central 1/3rd is not essential for infectivity. Genetically engineered λ phage variants contain restriction sites that flank the dispensable central third of genome. This segment may be cleaved by specific restriction enzyme and replaced be a foreign DNA segment of almost same length. The foreign DNA segment is annealed to the nicked phage DNA and ligated. Only those DNA segments that have length similar to the wild phages gets packaged into the heads. Those phages that have incorporated the “chimeric” chromosome become infectious.

Illustration on using λ phage Click to enlarge


Sometimes when human DNA is inserted into bacterial plasmid, it may not get expressed despite the presence of promoter. This is because bacteria RNA polymerase may not recognize promoter of human origin. This problem can be overcome by replacing human promoter region with bacterial promoter upstream of the gene. Such a vector containing bacterial promoter region (that result in expression of foreign gene) is called expression vector.

The DNA sequence that has been inserted into the vector is also called an “insert”.

Next: continuation of genetic engineering part 4 Transfer of chimeric DNA